The combination of two different electrophoresis methods has been known in many different forms and has been employed, for example, for separating part of the fractions from complex protein mixtures by means of a first electrophoresis step, and then to proceed with the separation by a second method, based on different parameters, in order to ensure that, for example, proteins that are irrelevant for the given problem will not influence the separation to be actually achieved, or in order to ensure in the case of complex protein mixtures that these are fractionated into all their different proteins, if possible, so as to obtain an overall map of the protein composition and to enable individual proteins to be detected.
A general summary of high-resolution 2D electrophoresis methods based on a polyacrylamide gel is provided by the paper by Michael J. Dunn and Arthur H. Burghes entitled "Review", published in the magazine "Electrophoresis 1983", 4, 97-116, so that there is no need to give a detailed description of these methods at this point; reference is made insofar to the before-mentioned publication and also to some additional publications corresponding to DE-OS 2,107,092; DE-OS 2,013,840; DE-PS 32,32,685 and/or the summary contained on page 31 of the book "Elektrohporese-Praktikum" by Dr. Rainer Westermeier, VCH Verlangsgesellschaft mbH, 6940 Weinheim. Reference is made to this book also with respect to the general understanding of electrophoretic separation processes, and the instruments, chemical substances and gels required for them.
In summary, in can be noted that for carrying out two-dimensional electrophoresis the methods most commonly employed to this day consist in using, for isoelectric focussing (separation after charging) in the first dimension, a gel having the largest possible pores and being different from the gel used for the second dimension, so that screening effects are avoided from the very beginning. In order to achieve the desired high reproducibility of the measured values, it is desirable that variations occurring when pouring the gel be possibly avoided so that it is preferred to make use of ready-to-use gels. This gives rise, however, to another problem because it is necessary in this case to introduce into the gel matrix what is known as chaotropic agents (urea) in order to avoid aggregates between proteins and complex formation, and because dissolved urea is chemically instable when such ready-to-use gels are to be stored.
It has also been known in this connection to use cylindrical gels, slab gels, round gels with nylon thread (millipore) or film-supported strips for separation in the first dimension.
Thereafter, the transfer from the first to the second dimension must be effected, for which purpose IEF spaghettis or IEF strips on carrier film, placed on vertical or horiziontal SDS gels, are suited best among all options available, with the resulting necessity to achieve reproducible re-equilibration in the SDS buffer, reprodicuble transfer and good contact between the first and the second dimension, and of course the need to check if all proteins have effectively migrated out from the first dimension.
For the second dimension, i.e. the separation by molecular weight (SDS electrophoresis) one normally uses a fine-pore gel having a high screening effect and a high pH value; but the high pH value gives ready-to-use gels a limited shelf life. If in this connection a discontinuous buffer system were used in order to achieve efficient separation, then--as is generally known--the buffers of ready-to-use gels would diffuse into each other during storage.
In connection with the problems arising with two-dimensional electrophoresis, reference is made finally to two recent publications, namely U.S. Pat. No. 4,874,490 (Hochstrasser) and the Japanese Patent No. JP 58105053 A2 (Hitachi).
The Hochstrasser patent uses two different gels for two-dimensional electrophoresis on a common carrier, for example a glass plate, namely a strip gel for the first dimenion and further a slab gel, the two gels being separated by an isolating area. The two gels are prepared ready for use, which means that they are wet gels, the strip gel containing in any case chaotropic agents and the slab gel being suited for carrying out SDS electrophoresis (SDS=sodium dodecyl sulfate).
For separation in the second dimension, the isolating area is removed in order to establish the necessary electric contact between the two gels. Consequently, there may arise in this case just the same problems as described before, namely instability of the ready-to-use gel containing the chaotropic agent, and in particular the necessity for the proteins to migrate into the slab gel via the previously isolating area. The Hochstrasser patent differs insofar only very little from the previously known, physically separate ready-to-use gels for two-dimensional electrophoresis, with the only difference that the first dimension is realized in geometric proximity to the second dimension.
In contrast to the above, the before-mentioned Japanese Patent JP 58105053 A2 proposes a rather complex and time-consuming method of carrying out two-dimensional separation, making use of a pore-gradient gel and doing without the usual SDS separation in the second dimension.
Consequently, the polyacrylamide matrix defined by its monomer concentration varies in concentration from, for example, between 4% and 20 or 25%, the lowest concentration that can be regarded as having particularly large pores being used for carrying out the separation in the first dimension in the usual way, by isoelectric focussing, while thereafter, as a result of the pore gradient variation, separation is effected by molecular size rather than by molecular weight. Another difficulty may present itself in connection with this two-dimensional separation method insofar as carrier ampholytes, i.e. so-called amphoteric buffer substances, each having different isoelectric points, must have been introduced into the entire gel for creating the necessary pH gradient.
Now, it is the object of the present invention to provide a high-resolution two-dimensional electrophoresis method which is carried out in a single gel and which can be realized with particular ease and at particularly low cost.